Cotton plant named 930 B Pima

ABSTRACT

The present invention relates to a method for rapidly introducing genes into germplasm which involves the use of crossing, backcrossing, intense selection and agronomic trait selection. More specifically, the method comprises the steps of: (a) crossing two parents of two different species, one of the parents (the donor parent) contains the gene of interest; (b) backcrossing the resultant progeny with the parent not containing the gene of interest (recurrent parent) for two to four backcrosses; (c) performing intense selection on a segregating generation of the progeny from the backcrossing step; and (d) selecting final germplasm on the basis of desired agronomic traits. The present invention further relates to two new and distinctive high yielding Pima Bt (Bacillus thuringiensis) Bollgard® cotton cultivars, designated 926 B Pima and 930 B Pima, which have been prepared in accordance with the method of the present invention.

This application is a divisional of U.S. patent application Ser. No.09/116,571 filed Jul. 16, 1998 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a method for rapidly introducing genesinto germplasm which involves the use of crossing, backcrossing, intenseselection and agronomic trait selection. More specifically, the methodcomprises the steps of: (a) crossing two parents of two differentspecies, one of the parents (the donor parent) contains the gene ofinterest; (b) backcrossing the resultant progeny with the parent notcontaining the gene of interest (recurrent parent) for two to fourbackcrosses; (c) performing intense selection on a segregatinggeneration of the progeny from the backcrossing step; and (d) selectingfinal germplasm on the basis of desired agronomic traits. The presentinvention further relates to two new and distinctive high yielding PimaBt (Bacillus thuringiensis) cotton cultivars, designated 926 B Pima and930 B Pima, which have been prepared in accordance with the method ofthe present invention.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.

Pureline cultivars, such as generally used in cotton and many othercrops, are commonly bred by hybridization of two or more parentsfollowed by selection. The complexity of inheritance, the breedingobjectives and the available resources influence the -breeding method.Pedigree breeding, recurrent selection breeding and backcross breedingare breeding methods commonly used in self pollinated crops such ascotton. These methods refer to the manner in which breeding pools orpopulations are made in order to combine desirable traits from two ormore cultivars or various broad-based sources. The procedures commonlyused for selection of desirable individuals or populations ofindividuals are called mass selection, plant-to-row selection and singleseed descent or modified single seed descent. One, or a combination ofthese selection methods, can be used in the development of a cultivarfrom a breeding population. Descriptions of breeding methods that arecommonly used for different traits and crops can be found in one ofseveral reference books (e.g., Allard, 1960; Welsh, 1981; Fehr, 1987;Mayo, 1987).

Pedigree breeding is primarily used to combine favorable genes into atotally new cultivar that is different in many traits than either parentused in the original cross. It is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁ (filial generation 1).An F₂ population is produced by natural selfing of plants or byphysically selling the plants. Selection of desirable individual plantsmay begin as early as the F₂ generation wherein maximum gene segregationoccurs. Individual plant selection can occur for one or moregenerations. Successively, seed from each selected plant can be plantedin individual, identified rows or hills, known as progeny rows orprogeny hills, to evaluate the line and to increase the seed quantity,or, to further select individual plants. Once a progeny row or progenyhill is selected as having desirable traits it becomes what is known asa breeding line that is specifically identifiable from other breedinglines that were derived from the same original population. At anadvanced generation (i.e., F₅ or higher) seed of individual lines areevaluated in replicated testing. At an advanced stage the best lines ora mixture of phenotypically similar lines from the same original crossare tested for potential release as new cultivars.

The single seed descent procedure in the strict sense refers to plantinga segregating population, harvesting one seed from every plant, andcombining these seeds into a bulk which is planted the next generation.When the population has been advanced to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. Primary advantages of the seed descentprocedures are to delay selection until a high level of homozygosity(e.g., lack of gene segregation) is achieved in individual plants, andto move through these early generations quickly, usually through usingwinter nurseries.

The modified single seed descent procedures involve harvesting multipleseed (i.e., a single lock or a simple boll) from each plant in apopulation and combining them to form a bulk. Part of the bulk is usedto plant the next generation and part is put in reserve. This procedurehas been used to save labor at harvest and to maintain adequate seedquantities of the population.

Backcrossing is the preferred method of transferring one or more simplyinherited genes from one variety (the donor parent) to a second variety(the recurrent parent). The F₁ is continually crossed with the recurrentparent until the recurrent parent is recovered. Backcrossing is simple,requires small plant populations and generates results quickly. Theaccumulation of genes from the recurrent parent increases at a rate of50% with each backcross.

The number of backcrosses required to recover the recurrent parentincreases as the genetic distance between the donor and recurrent parentdiverge. Backcrosses involving interspecific parents represent extremegenetic distance. Deleterious genetic interactions and even problems atthe chromosome level are common. Numerous backcrosses are necessary torecover the recurrent parent.

The recovery of the recurrent parent is based on the probability ofmoving closer to that parent by 50% with each backcross, randomly. Whendeleterious genetic interactions occur, even with 99.99% of the genomeof the recurrent parent reclaimed, it may not be sufficient. A gene ormany genes responsible for the deleterious genetic interactions may bestill present in the 0.01% of the donor parent DNA. Insignificant ratiosof recurrent parent/donor parent DNA can have significant consequencesin plant genomes.

Selection for desirable traits can occur at any segregating generation(F₂ and above). Selection pressure is exerted on a population by growingthe population in an environment where the desired trait is maximallyexpressed and the individuals or lines possessing the trait can beidentified. For instance, selection can occur for disease resistancewhen the plants or lines are grown in natural or artificially-induceddisease environments, and the breeder selects only those individualshaving little or no disease and are thus assumed to be resistant.

Promising advanced breeding lines are thoroughly tested and compared topopular cultivars in environments representative of the commercialtarget area(s) for three or more years. The best lines havingsuperiority over the popular cultivars are candidates to become newcommercial cultivars. Those lines still deficient in a few traits arediscarded or utilized as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take from seven to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior because, for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental lines and widely grown standardcultivars. For many traits a single observation is inconclusive, andreplicated observations over time and space are required to provide agood estimate of a line's genetic worth.

The two cotton species commercially grown in the United States areGossypium hirsutum, commonly known as short staple or upland cotton andGossypium barbadense, commonly known as extra long staple (ELS) or, inthe United States, as Pima cotton. Upland cotton fiber is used in a widearray of coarser spin count products. Pima cotton is used in finer spincount yarns (50-80) which are primarily used in more expensive garments.Other properties of Pima cotton are critical because of fiber end use.

Cotton is an important and valuable field crop. Thus, a continuing goalof plant breeders is to develop stable, high yielding cotton cultivarsthat are agronomically sound. The reasons for this goal are obviously tomaximize the amount and quality of the fiber produced on the land usedand to supply fiber, oil and food for animals and humans. To accomplishthis goal, the cotton breeder must select and develop plants that havethe traits that result in superior cultivars.

G. barbadense is a cultivated species of cotton denoted by the genome(AD)2. Peru is the proximate area considered to be the center of originfor the species. G. barbadense varieties grown in the United States arecommonly referred to as "American Pima", Pima or ELS cottons. The terms"American Pima", Pima and ELS are elucidated in the Definitions listedbelow.

It is often desired to introduce traits from one cotton species into theother. Such interspecific crosses are generally hard to work withbecause the chromosomes of the distinct species involved do not pairwell in the progeny. The consequence is a large number of progeny thatare very poor relative to either parent post F₁ generation.Interspecific crosses between upland and Pima species, where selectiontoward the Pima parent is desired, is further complicated by the factthat Pima fiber traits are held to very high standards.

The goal of a commercial cotton breeding program is to develop new,unique and superior cotton cultivars. In cotton, the important traitsinclude higher fiber (lint) yield, earlier maturity, improved fiberquality, resistance to diseases and insects, tolerance to drought andheat, and improved agronomic traits. The breeder initially selects andcrosses two or more parental lines, followed by generation advancementand selection, thus producing many new genetic combinations. The breedercan theoretically generate billions of different genetic combinationsvia this procedure. The breeder has no direct control over which geneticcombinations will arise in the limited population size which is grown.Therefore, two breeders will never develop the same line having the sametraits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The lines whichare developed are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce, with any reasonable likelihood, thesame cultivar twice by using the exact same original parents and thesame selection techniques. This unpredictability results in theexpenditure of large amounts of research moneys to develop superior newcotton cultivars.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, and the grower, processor and consumer; forspecial advertising and marketing and commercial production practices,and new product utilization. The testing preceding the release of a newcultivar should take into consideration research and development costsas well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

The development of new cotton cultivars requires the evaluation andselection of parents and the crossing of these parents. The lack ofpredictable success of a given cross requires that a breeder, in anygiven year, make several crosses with the same or different breedingobjectives.

The cotton flower is monecious in that the male and female structuresare in the same flower. The crossed or hybrid seed is produced by manualcrosses between selected parents. Floral buds of the parent that is tobe the female are emasculated prior to the opening of the flower bymanual removal of the male anthers. At flowering, the pollen fromflowers of the parent plants designated as male, are manually placed onthe stigma of the previous emasculated flower. Seed developed from thecross is known as first generation (F₁) hybrid seed. Planting of thisseed produces F₁ hybrid plants of which half their genetic component isfrom the female parent and half from the male parent. Segregation ofgenes begins at meiosis thus producing second generation (F₂) seed.Assuming multiple genetic differences between the original parents, eachF₂ seed has a unique combination of genes.

There is a need in the art to increase the efficiency of breeding newcultivars having agronomically improved traits, especially new cultivarsderived from interspecies. There is also a need in the art to developnew cultivars of Pima cotton which contain the Bt gene. The presentinvention describes a method for rapidly introducing genes intogermplasm, such as the Bt gene (Bollgard®) into Pima cotton, and thussatisfies these needs.

SUMMARY OF THE INVENTION

The present invention relates to a method for rapidly introducing genesinto germplasm which involves the use of crossing, backcrossing, intenseselection and agronomic trait selection. More specifically, the methodcomprises the steps of: (a) crossing two parents of two differentspecies, one of the parents (the donor parent) contains the gene ofinterest; (b) backcrossing the resultant progeny with the parent notcontaining the gene of interest (recurrent parent) for two to fourbackcrosses; (c) performing intense selection on a segregatinggeneration of the progeny from the backcrossing step; and (d) selectingfinal germplasm on the basis of desired agronomic traits. The method ofthe present invention is particularly adapted to moving genes or traitsfrom one species into a second species.

The present invention further relates to two new and distinctive highyielding Pima Bt (Bacilus thuringiensis) Bollgard® cotton cultivars,designated 926 B Pima and 930 B Pima, which have been prepared inaccordance with the method of the present invention. The presentinvention also relates to the seeds of these cotton cultivars, to theplants of these cotton cultivars and to methods for producing a cottonplant by crossing each of these cotton cultivars with itself or anothercotton line.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for rapidly introducing genesinto germplasm which involves the use of crossing, backcrossing, andintense selection for superior product quality and agronomic traits.More specifically, the method comprises the steps of: (a) crossing twoparents of two different species, one of the parents (the donor parent)contains the gene of interest; (b) backcrossing the resultant progenywith the parent not containing the gene of interest (recurrent parent)for two to four backcrosses; c performing intense selection on asegregating generation of the progeny from the backcrossing step; and(d) selecting final germplasm on the basis of desired agronomic traits.The method of the present invention is particularly adapted to movinggenes or traits from one species into a second species.

The present invention further relates to two new and distinctive highyielding Pima Bt (Bacillus thuringiensis) Bollgard® cotton cultivars,designated 926 B Pima and 930 B Pima, which have been prepared inaccordance with the method of the present invention. The presentinvention also relates to the seeds of these cotton cultivars, to theplants of these cotton cultivars and to methods for producing a cottonplant by crossing each of these cotton cultivars with itself or anothercotton line.

In the development of new varieties, especially involving interspeciescrosses, in which backcrossing with a recurrent parent is the standardtechnique, it is conventional wisdom that a minimum of 6 and morepreferably 7-10 backcrosses are necessary to introduce a gene from thedonor parent into the recurrent parent. It has been discovered, inaccordance with the present invention, that the number of backcrossescan be significantly reduced, e.g. to two to four backcrosses, byapplying intense selection to the progeny of the final backcross (i.e.,to the progeny of the second, third or fourth backcross), followed byselection for desired agronomic characteristics of the progeny of theintense selection. This discovery was totally unexpected and now enablesthe rapid production of new varieties using a backcrossing breedingtechnique.

Before the method of the present invention is described, the followingdefinitions are provided to provide a clear and consistent understandingof the present invention.

DEFINITIONS

As used herein the term "American Pima" describes a sub-set of Gossypiumbarbadense cotton varieties developed in the desert southwest startingin the early 1900's. In 1908 the USDA moved it's G. barbadense breedingprogram to the Gila River Pima Indian reservation at Sacaton, Ariz.

Current American Pima cottons trace their background to a complex set ofcrosses involving the G. barbadense cottons Sea Island, Pima (anindirect selection from the Egyptian variety Mit Afifi), Tangus and theG. hirsutum variety Stoneville.

American Pima varieties are grown because of superior fiber qualitiesthat command a monetary premium relative to G. hirsutum fiber that islong, strong, uniform and fine but mature. The fiber is used to spinvery high quality yarns that contain cotton.

"BC_(n) " refers to the nth backcross with the recurrent parent. Forexample, the first backcross of the recurrent parent with the F_(i) ofthe initial cross is BC₁ ; the second backcross of the progeny of thefirst backcross is BC₂, etc.

"BC_(n) F_(m) " refers to the mth generation of the nth backcross. Forexample, BC₃ F₁ is the progeny (seeds) of the third backcross; BC₃ F₂ isthe progeny of plants grown from the seeds of BC₃ F₁, etc. BC₃ F₁ andBC₃ can be used interchangeably.

The term "Desired Agronomic Characteristics" refers to agronomiccharacteristics (which will vary from crop to crop and plant to plant)such as yield, maturity, pest resistance and lint percent which aredesired in a commercially acceptable crop or plant. For example,improved agronomic characteristics for cotton include yield, maturity,fiber content and fiber qualities.

"Donor Parent" refers to the parent of a variety which contains the geneor trait of interest which is desired to be introduced into a secondvariety.

"ELS" is the abbreviation for "Extra Long Staple". ELS is the groupclassification for cottons in the longest staple length category. Asused in practice and for commerce, ELS denotes varieties belonging tothe species G. barbadense that have superior fiber qualities, includingclassification in the longest staple length category.

The term "Fiber Characteristics" refers to fiber strength, fiber length,micronaire, fiber elongation and uniformity of fiber.

The term "Fiber Elongation" also sometimes referred to as "E1" refers tothe elongation of the fiber at the point of breakage in the strengthdetermination.

The term "2.5% Fiber Span Length" refers to the longest 2.5% of a bundleof fibers expressed in inches as measured by a digital fibergraph.

The term "Fiber Strength" also sometimes referred to as "T1" refers tothe force required to break a bundle of fibers. Fiber strength isexpressed in millinewtons (mn) per tex on a stelometer.

"Fruiting Nodes" are the number of nodes on the main stem from whicharise branches that bear fruit or boll in the first position.

The term "Gin Turnout" refers to fraction of lint in a machine harvestedsample of seed cotton (lint, seed, and trash).

The term "Intense Selection" as used herein, refers to the degree ofselection applied to the progeny of a backcross and means that thecriteria for the selection is that (a) most or all of the traits orcharacteristics of the recurrent parent are selected for and (b) most orall of the traits or characteristics of the donor parent other than thegene of interest are selected against. If there are no progeny that meetthe full criteria after selecting for the recurrent parent traits, thenthe progeny that most closely fit the criteria are selected, followed bypedigree selection and a second generation of selection.

The term "Lint Yield" refer to the measure of the quantity of fiberproduced on a given unit of land. Presented below in pounds of lint peracre.

"Lint Percent" refers to the lint (fiber) fraction of seed cotton (lintand seed).

The term "Maturity Rating" means a visual rating near harvest on theamount of open boils on the plant. The rating range is from 1 to 5, 1being early and 5 being late.

"Micronaire" refers to a measure of the fineness of the fiber. Within acotton cultivar, micronaire is also a measure of maturity. Micronairedifferences are governed by changes in perimeter or in cell wallthickness, or by changes in both. Within a variety, cotton perimeter isfairly consistent and maturity will cause a change in micronaire.Consequently, micronaire has a high correlation with maturity within avariety of cotton. Maturity is the degree of development of cell wallthickness. Micronaire may not have a good correlation with maturitybetween varieties of cotton having different fiber perimeter. Micronairevalues range from about 2.0 to 6.0:

    ______________________________________                                        Below 2.9                                                                             Very fine                                                                              Possible small perimeter but mature                                           (good fiber), or large perimeter                                              but immature (bad fiber).                                    2.9 to 3.7                                                                            Fine     Various degrees of maturity and/or perimeter.                3.8 to 4.2                                                                            Average  Average degree of maturity and/or perimeter.                 4.3 & above                                                                           Coarse   Usually fully developed (mature),                                             but larger perimeter.                                        ______________________________________                                    

The term "Pima" is used in the same context as "American Pima". Note,the term "Peruvian Pima" is used to denote varieties developed in Perufrom the American variety "Pima".

"Plant Height" refers to the average height in meters of a group ofplants.

The term "Recurrent Parent" refers to the repeating parent (variety) ina backcross breeding program. The recurrent parent is the variety intowhich a gene or trait is desired to be introduced.

The term "Fiber Span Length" refers to the distance spanned by aspecific percentage of fibers in a test specimen, where the initialstarting point of the scanning in the test is considered 100 percent asmeasured by a digital fibergraph.

"Stringout Rating" also sometimes referred to as "Storm Resistance"refers to a visual rating prior to harvest of the relative looseness ofthe seed cotton held in the boll structure on the plant. The ratingvalues are from 1 to 5 (tight to loose in the boll).

The term "Uniformity Ratio" refers to a measure of the relative fiberspan length uniformity of a bundle of fibers. The uniformity ratio isdetermined by dividing the 50% fiber span length by the 2.5% fiber spanlength.

"Vegetative Nodes" are the number of nodes from the cotyledonary node tothe first fruiting branch on the main stem of the plant.

In accordance with the present invention, a method is provided forrapidly introducing a gene or trait from one variety (a donor parent)into a second variety (a recurrent parent). The objectives of thepresent invention are twofold. The first objective is to move thetransgene(s) to the recurrent parent phenotype. The second objective isto accomplish the first objective as quickly as genetically possibleusing minimal backcrossing generations in conjunction with intenseselection. The method is particularly well adapted for introducing agene or trait from one species (a donor parent) into a second species (arecurrent parent).

Generally, the present method comprises the steps of: (a) crossing adonor parent with a recurrent parent; (b) backcrossing the resultingprogeny with the recurrent parent for two to four backcrosses; and (c)performing intense selection on a segregating generation of the progenyof the backcrossing step. In a preferred embodiment the method furthercomprises the step of: (d) selecting a final variety on the basis ofdesired agronomic characteristics. The donor parent contains the gene ortrait of interest which can be any desired gene or trait. Intenseselection is performed by selecting for traits and characteristics ofthe recurrent parent and selecting against any trait or characteristicof the donor parent In a preferred embodiment intense selection alsoincludes selecting for full expression of the gene or trait of interest.

More specifically, the method of the present invention comprises thesteps of: (a) crossing said donor variety with said recurrent variety toproduce a first familial generation variety; (b) crossing said recurrentvariety with said first familial generation variety to produce a firstbackcross-first familial generation variety. In backcrosses involvinggenetically diverse recurrent and donor parents selection of BC₁ F₁plants which are most similar to the recurrent parent should be selectedas the donor parent in the immediate subsequent backcross; (c) crossingsaid recurrent variety with said first backcross-first familialgeneration variety to produce a second backcross-first familialgeneration variety; and (d) subjecting a segregating generation of saidsecond backcross-first familial generation variety to intense selectionto produce a selected variety having the characteristics of saidrecurrent variety and the gene or trait of said donor variety. In apreferred embodiment the method further comprises the step of: (e)subjecting the selected variety to further selection for desiredagronomic characteristics. In a preferred embodiment, the intenseselection is performed on the second or third segregating generation ofthe progeny of step (c), i.e., BC₂ F₃ or BC2F₄.

In a second embodiment of the method of the present invention, anadditional backcrossing step is incorporated between steps (c) and (d).According to this embodiment, the method further comprises the step of:(cl) crossing said recurrent variety with said second backcross-firstfamilial generation variety to produce a third backcross-first familialgeneration variety. A segregating generation of said thirdbackcross-first familial generation variety is subjected to the intenseselection of step (d). In a preferred embodiment, the intense selectionis performed on the second or third segregating generation of theprogeny of step (cl), i.e., BC₃ F₃ or BC₃ F₄.

In a third embodiment of the method of the present invention, anadditional backcrossing step is incorporated between steps (cl) and (d).According to this embodiment, the method further comprises the step of:(c2) crossing said recurrent variety with said third backcross-firstfamilial generation variety to produce a fourth backcross-first familialgeneration variety. A segregating generation of said fourthbackcross-first familial generation variety is subjected to the intenseselection of step (d). In a preferred embodiment, the intense selectionis performed on the second or third segregating generation of theprogeny of step (c2), i.e., BC₄ F₃ or BC₄ F₄.

The present invention is further directed to novel varieties of Pimacotton which contain a Bollgard® gene. The Bt gene is the Bollgard® geneconstruct 531 which has been inserted into upland cotton. Specifically,the invention is directed to the novel cultivars designated 926 B Pimaand 930 B Pima. The invention is further directed to methods ofproducing these cultivars as well as to methods of using the cultivarsand plants and plant parts of the cultivars. Methods of using thecultivars include crossing a first parent cotton plant with a secondparent cotton plant, wherein the first or second cotton plant is thecotton plant from the designated cultivars. Further, both first andsecond parent cotton plants may be from the same designated cultivar.Therefore, any methods using the cultivars of the present invention arepart of this invention: selfing, backcrosses, hybrid breeding, andcrosses to populations. Any plants produced using the present cultivarsas a parent are within the scope of this invention. As used herein, theterm "plant" includes plant cells, plant protoplasts, plant cells oftissue culture from which cotton plants can be regenerated, plant calli,plant clumps, and plant cells that are intact in plants or parts ofplants, such as pollen, flowers, seeds, pods, leaves, stems, and thelike. Thus, another aspect of this invention is to provide for cellswhich upon growth and differentiation produce the designated cultivars.Trolinder, N. L., et al., Somatic embryogenesis and plant regenerationin cotton, Plant Cell Reports 6:231-234 (1987); Bailey, C., et al.,Engineering 2, 4-D resistance into cotton, Theoretical and AppliedGenetics 83:645-649 (1992).

The method of the present invention will be further illustrated withreference to cotton, and specifically with reference to Gossypsiumhirsutum and Gossypium barbadense, and the Bacillus thuringiensis geneas the gene of interest. It is understood that the present method isequally applicable to any other agronomic or horticultural plant and toany other gene or trait of interest.

As previously discussed, the method involves a step of intense selectionin which the traits of the recurrent parent, Pima (G. barbadense), inthis example are selected for, and any trait of the donor parent, upland(G. hirsutum), in this example are selected against. The phenotypicdifferences between upland and Pima cotton are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Phenotypic differences between Pima (G. barbadense)                           and Delta type Upland (G. hirsutum) cottons.                                  Traits    Pima (G. barbadense)                                                                          Upland (G. hirsutum)                                ______________________________________                                        Leaves    Long leaf lobes 2/3-cut                                                                       Weak leaf lobes 1/2-cut                                                       or less                                                       Green           Olive                                                         Shiny           Less shiny                                                    One to five foliar nectaries                                                                  One to three foliar                                           on underside    nectaries on underside                                        Leaf lobes slightly                                                                           Leaf lobes not                                                constricted at base                                                                           constricted at base                                           Sinuses usually thrown up                                                                     Sinuses not or only                                           in folds        slightly overlapping                                Stipules  10-50 mm long   4-15 mm long                                        Brateoles Almost as broad as wide                                                                       Longer than broad                                             Teeth 10-15 mm long                                                                           Teeth 7-12 mm long                                  Calyx     6-10 mm long    5-6 mm long                                         Petals    Up to 80 mm long                                                                              Up to 50 mm long                                              Yellow          Cream to pale yellow                                          Dark red leaf spot at base                                                                    Leaf spot absent                                    Style     Exceeds androccium                                                                            More or less enclosed by                                                      androccium or somewhat                                                        exceeds androccium                                  Stamatal column                                                                         Long            Short                                               Stigma    Cleft near tip predominate                                                                    United throughout                                             in first flowers of the                                                       growing season, thereafter                                                    occurs occasionally                                                 Pollen    Yellow          Cream to pale yellow                                                          (in specific upland used)                           Capsules  Three celled    Four to five celled                                           Prominently pitted                                                                            Slightly pitted                                               Usually somewhat                                                                              Broadly ovoid or                                              elongated       subglobasose                                        Seed      Naked seed, seed fuzz                                                                         Thick coat of seed fuzz                                       absent or tufted ends                                               Fiber properties                                                              (relative)                                                                    25% span length                                                                         25% longer than upland                                                                        20% shorter than Pima                               Uniformity                                                                              7% higher uniformity than                                                                     6% lower uniformity                                           Upland          than Pima                                           Micronaire                                                                              7% lower micronaire than                                                                      7% higher micronaire                                          Upland          than Pima                                           Strength  56% higher strength than                                                                      36% lower strength                                            Upland          than Pima                                           ______________________________________                                    

A cross is made between an agronomic Pima parent (recurrent parent) andan upland transgene(s) donor. The resulting F₁ seed is backcrossed tothe recurrent parent. A larger number of crosses to the F₁ are maderelative to conventional backcrossing. The amount of seed needed isincreased because selection for the transgene(s) and the Pima parentwill be exerted on the BC₁ plant population. A dominant transgene (i.e.Bt gene) segregates one to one for plants containing the Bt gene.Therefore, two times the number of plants needed to select for the Pimaplant type are required. If two dominant transgenes are beingtransferred, four times the number of plants needed to select for thePima plant type are required to recover both transgenes simultaneously.

The BC₁ seed (also referred to as (BC₁ F₁ seed) is planted at a nurserywhere the plants (BC₁ F₁) can be closely monitored, if desired. It ispossible with the present invention to make the backcrosses without anyselection. Segregation occurs as a result of the initial Pima by uplandcross. All traits expressed represent homozygous loci donated by thePima parent or heterozygous loci with an allele donated by both speciesused in the cross. Many identifiable heterozygous qualitativelyinherited traits are observable. It is seen that variability betweenselected plants exists. In genetically narrow crosses this variabilitycan be exploited by intercrossing. The objective is to proceed with aconventional breeding program within the population. As progenyhomozygous for the transgene are identified and intercrossed, the needfor extensive testing for the transgene(s) at every step is eliminated,providing the opportunity to concentrate on agronomic improvement.Alternatively, an initial selection for the transgene(s) can be exertedon the BC₁ plant population. Plants containing the transgene(s) ofinterest can then be used for further backcrossing. Although additionalscreening is not necessary at this stage for the operation of thepresent invention, progeny of plants with the most homozygous traits canbe used in one embodiment for the BC₂ cross. Backcrossing to selectedBC₁ F₁ 's reduces the number of undesirable G. hirsutum chromosomes farmore effectively than random elimination expected through a backcrossalone.

Again, a relatively large BC₂ F₁ population from the BC₂ backcross isplanted, selected for the transgene(s), if desired and self pollinated.A very large BC₂ F₂ population from the selected BC₂ F₁ population isplanted, subjected to intense selection which can include selection forthe transgene(s). In the present description for cotton, the traits ofG. barbadense selected for and the traits of G. hirsutum selectedagainst are shown in Table 2. The selected BC₂ F₂ plants are then selfpollinated.

                  TABLE 2                                                         ______________________________________                                        Traits for Selection                                                          ______________________________________                                        Selected Against    Selected For                                              olive plant color   bright green plant color                                  reduced leaf lobe   deep leaf lobe                                            cream colored flower petal                                                                        full yellow colored flower                                                    petal                                                     absent of no flower petal spot                                                                    petal spot presence                                       cream/intermediate pollen color                                                                   yellow pollen color                                       locule numbers other than three                                                                   three locule bolls                                        small gossypol glands                                                                             larger gossypol glands                                    shallow boll pitting                                                                              deep boll pitting                                         *inferior fiber quality                                                                           *pima fiber quality                                       (length < 1.30,     (length >1.30,                                            strength < 32.5,    strength > 32.5,                                          mic > 4.0)          mic around 4.0)                                           <100% expression of the gene of interest                                                          100% expression of the                                    from the donor parent                                                                             gene of interest from                                                         the donor parent                                          upland plant type   pima plant type                                           fuzzy seed (lint clings to seed coat)                                                             naked seed                                                                    (lint does not cling                                                          to seed coat)                                             ______________________________________                                         *fiber quality based on hand pulling and rating the fiber in the field.  

BC₂ F₃ seed from BC₂ F₂ plant selections, possessing the transgene(s)are planted to row. Within selected rows plant selections are made forthe recurrent parent plant type and desired agronomic characteristics,such as yield and fiber properties. Some breeders may choose to plantprogeny rows and identify homozygous rows in season, i.e., herbicideresistance.

BC₂ F₄ seed from BC₂ F₃ plant selections homozygous for the transgene(s)are planted to row and selected for the recurrent parent plant type anddesired agronomic characteristics, such as yield and fiber properties.Individual rows are bulk harvested and increased for replicated yieldtests or plant selections can be made as in normal pedigree breeding.

The previous description related to the development of a Pima plant typecontaining the transgene(s) at the BC₂ F₄ generation. In a similarmanner, a Pima plant type containing a transgene of interest can bedeveloped at the BC₄ F₄ generation. As in the previous description, across is made between an agronomic Pima parent (recurrent parent) and anupland transgene donor. Conventional backcrossing is used through theBC₂. A large population of the progeny of the BC can be used withoutselection or selection can be based on the transgene.

The BC₂ seed is planted at a nursery where the plants can be closelymonitored, if desired. For this example, selection for the transgene(s)is exerted on the BC₂ F₁ plant population. The selected progeny is thenbackcrossed with either the recurrent parent, or it can be crossed withanother superior Pima variety to direct the population genetics desiredby the breeder.

A population breeding program can be initiated at this point to createvariability within the backcross population. The object of increasingthe variability is to develop a population that a number of Pimavarieties with the transgene(s) of interest can be selected from over anumber of years. It is also possible the transgene(s) will function moreefficiently in certain Pima genetic backgrounds. If this is true, theprobability of identifying those backgrounds will increase.

Seed from the BC crosses are handled as the BC₂ population above. It maybe possible to select desirable agronomic phenotypes resulting fromincreased variability in the Pima genetic background.

Seed from the BC₄ crosses are handled as the BC₂ population above. Therecurrent genotypes or other genotypes used for breeding should consistof as wide an array as possible of superior Pima varieties to createvariability.

Again, a relatively large BC₄ F₁ population from the BC₄ backcross isplanted, selected for the transgene(s), if desired and self pollinated.A very large BC₄ F₂ population from the selected BC₄ F₁ population isplanted, subjected to intense selection which can include selection forthe transgene(s). The traits for intense selection are set forth inTable 2. The selected BC₄ F₂ plants are then self pollinated. Careshould be taken not to narrow the Pima genetic base through severeselection for specific Pima phenotypes. Alternatively, the BC₄ F₂population is self-fertilized. The BC₄ F₃ plants are then subjected tothe intense selection.

BC₄ F₃ seed from BC₄ F₂ plant selections homozygous for the transgene(s)are planted to row. Within selected rows plant selections are made forthe recurrent parent plant type and desired agronomic characteristics,such as yield and fiber properties. Alternatively, if intense selectionis performed on the BC₄ F₃ plants, the BC₄ F₄ seed is used at thisstage.

BC₄ F₄ seed from BC₄ F₃ plant selections (or BC F ₅ seed from BC₄ F₄plant selections in the alternative) homozygous for the transgene(s) areplanted to row. Individual rows are bulk harvested and increased forreplicated yield tests. If advantageous, individual plants can beselected for pedigree breeding.

In accordance with the method of the present invention, the Bollgard®gene (a Bt gene) is moved from upland (G. hirsutum) to Pima S6 (G.barbadense) cotton. Utilizing upland cotton as the Bollgard® gene donorand Pima S6 as the recurrent parent exemplifies extensive geneticdistance. Interspecific crosses approach the boundary of transfer ofgenetic material through sexual reproduction in higher plants.Conventionally, seven or more backcrosses is the minimum number ofbackcrosses accepted as needed to fully recover the Pima S6 recurrentparent phenotype. However, as shown in further detail herein, a Pima S6recurrent parent phenotype having the Bollgard® gene is recovered withonly three backcrosses.

Initially, the Bollgard® donor (e.g., Coker 312) is crossed with therecurrent Pima S6 parent. The F₁ is backcrossed with Pima S6. Theprogeny of the BC₁ backcross is again backcrossed with Pima S6, and theprogeny of the BC₂ backcross is backcrossed with Pima S6. No selectionsare made in the course of the BC₁, BC₂ or BC₃ backcrosses. Progeny ofthe BC₃ backcross are self-pollinated for two generations to produceseed from the BC₃ F₂ plants. The seed is planted and the BC₃ F₃ plantsare subjected to intense selection on the basis of the traits set forthin Table 2, including Pima fiber and Bollgard® gene expression. Theplant rows are graded for yield and the best plants within the selectedrows are harvested. Plant selections from BC₃ F₃ are planted to row andthe highest yielding, true breeding rows with Pima phenotype andhomozygous for the Bollgard® gene are bulk harvested. BC₃ F₃ derivedlines are tested in two replicated tests, increased and bulk harvestedwithout selection.

As discussed, selection is initiated in the BC₃ F₃ generation (1995).Individual lines identified as homozygous for the Bollgard® gene areplanted. Based on probabilities associated with backcrossing, genes fromthe Pima recurrent parent represented 93.75% of the genes in thepopulation. The explosion of phenotypic variability caused by genescontributed by both species as they became homozygous is amazing.Because the BC₃ F₃ population is segregating for a large number ofphenotypic traits readily identifiable as belonging to G. hirsutum or G.barbadense tremendous selection pressure could be applied. The largenumber of segregating traits is the critical component that made theunanticipated success of recovering Pima S6 plant type with theBollgard® gene possible. The full recovery of Pima S6 plant type isunexpected at backcross three.

For the intense selection, all plants that exhibited any trait derivedfrom G. hirsutum was eliminated, with the exception of the Bollgard®gene. The backcross involved interspecific parents and an earlybackcross generation, recombination between chromosomes of theindividual species were relatively infrequent. The species specifictraits identified a segment of DNA possibly as large as entirechromosomes. Enormous quantities of genetic material belonging to G.barbadense is accumulated while eliminating G. hirsutum genetic materialin the selected plants. The expressed G. hirsutum and G. barbadensetraits facilitated precise and very effective selection for genescontributed by the recurrent parent, Pima S6. In contrast, conventionalbackcrossing is based on progressive but random accumulation of geneticmaterial from the recurrent parent.

Rows are used to judge yield for two generations. Plant selections areconsidered exclusively in the highest yielding rows, homozygous for theBollgard® gene. Yield is obviously important for commercial reasons.Yield also identifies lines containing genetic material that functionswell together in the genome, regardless of species origin. Antagonisticgene interactions and problems at the chromosome level are common ininterspecific crosses.

The suitability of the present invention is seen in comparing the BC₃ F₄plant selections with lines derived from additional backcrosses inaccordance with conventional techniques. For this comparison, BC₆ F₃rows are planted in the same field as the BC₃ F₄ plant selections. TheBC₆ F₃ rows expressed approximately 10% G. hirsutum traits than the BC₃F₄ rows. Fertility was reduced in the BC₆ F₃ rows as evidenced by 2 to 4seed per lock compared to 6 to 7 seed per lock in the BC₃ F₄ rows. TheBC₆ F₃ material requires another backcross with selection to alleviatethe fertility problems.

Plant selections from the BC₃ F₃ planted the following year were free ofreadily identifiable traits introduced by the donor parent, with theexception of the Bollgard® gene. Plant and row selections were madeexclusively on the quantitatively inherited traits yield, fiberproperties and maturity. Four experimental strains derived from singleplant selections from one BC₃ F₃ row remain in testing. Two of the fourremaining experimental strains are likely to be discarded because theyare excessively late maturing. Based on plant selection, the selectionindex is 0.00056. Based on row selection, the selection index is 0.0041.

It is believed that three reasons contribute to the recovery of Pima S6plant type with the Bollgard® gene where intense selection was initiatedat the BC₃ F₃ generation from an interspecific cross. First, massivequantities of identifiable, qualitative and quantitative, speciesspecific genetic traits were generated as a result of interspecificgenetic exchange. In many cases, intermediate expression of qualitativegenes identified heterozygous plants. The traits were used to select forthe G. barbadense parent and against the G. hirsutum parent. The haploidchromosome number in both species is 26. If no crossovers is assumed,and each chromosome is identified by one trait, 26 species specifictraits are required to identify each chromosome and donor species.Crossovers did occur and one to many traits may have identified the samechromosome. The quantity of species specific traits is the primaryreason for success.

Second, recombination was minimized. Recombination is reduced ininterspecific exchanges. The chromosomes of the species involved arephysically different, but are similar enough to pair. Initiation ofselection in the BC₃ F₃ reduced opportunities for recombination inhigher backcross generations. Reduced recombination means on average,large species specific linkage groups were identified by the speciesspecific genetic traits.

Third, extreme selection pressure was exerted on the BC₃ F₃ plants androws. Both qualitative and quantitative traits were used to identifyPima S6 plant type. All rows deficient in yield or fiber wereeliminated. Within the selected rows only plants expressing traitsinherited exclusively from the Pima S6 parent were selected. Allselected plants were fiber tested for Pima fiber quality, all deficientplant selections were eliminated.

One very surprising development of this selection methodology is thesuperior fiber quality recovered in the initial selections. Fiberquality is critical in Pima cotton. All fiber traits are inheritedquantitatively and very difficult to recover after interspecificcrosses. The quick recovery of Pima fiber quality supports thehypothesis that significant quantities of upland genes were eliminatedfrom the selected population by the BC₃ F₄ generation when extremeselection pressure was applied to it.

The present invention is described by reference to the followingExamples, which is offered by way of illustration and is not intended tolimit the invention in any manner. The Examples describe the productionof two novel Pima cotton varieties with the Bollgard® trait produced inaccordance with the present invention.

EXAMPLE 1 Pima Cotton Cultivar 926 B Pima

Pima cotton cultivar 926 B Pima has superior characteristics and wasdeveloped in accordance with the method of the present invention fromthe cross of upland cotton Coker 312 (the donor parent) to Pima S6 (therecurrent parent). Pima S6 is a USDA released public variety.Backcrossing was used initially to transfer the Bt gene into the newvariety. The initial breeding was accomplished in greenhouses from thestart of the project through BC₃ F₂.

In the spring of 1995, seed from 241 individual BC₃ F₂ plants developedat Scott, Mississippi were sent to Casa Grande, Ariz. to be planted inthe field for evaluation. Prior to planting, 10 seeds from eachindividual line were evaluated for Bt expression in the seed. The objectwas to identify the lines homozygous for the Bt gene. 65 BC₃ F₃ tested100% for the Bt gene and were planted in the field in 1995 [for aselection pressure of 0.27 (65/241) or 27%]. Seed from each line wasplanted in as many rows as possible and thinned to one plant per foot tomaximize the number of plants for selection.

Throughout the 1995 growing season plants exhibiting any upland cottontraits were tagged so they could be avoided at harvest. Examples oftraits, against which selection was conducted, are light green plantcolor, reduced leaf lobe, cream flower petal color, absent or no flowerpetal spot, cream/intermediate pollen color, locule numbers differentthan three, small gossypol glands and shallow boll pitting.

In June, 1995 ten plants from each row were sampled for Bt expression inplant tissue. It was observed that some plants exhibited weakexpression. The reason for this is not known. It may be the testingtechnique or true weak expression of the gene in specific genotypes. Anyrow expressing less than 100% Bt was discarded. Strong negativeselection pressure was also placed on rows weakly expressing the Bt gene[for a selection pressure of 0.149 (36/241) or 14.9%].

At harvest each row was visually graded for yield. Once the best lineswere identified each plant in the selected line was examined by hand forfiber quality and naked seed (lint does not cling to the seed coat).Substandard plants were discarded [for a row selection pressure of 0.05(12/241) or plant selection pressure of 0.033 or 3.3%].

Selected plants were ginned and the seed was examined for nakedness.Lines considered too fuzzy were discarded. The fiber of the remainingplants tested with standard instruments. All selections exhibitingsubstandard fiber were discarded. Twenty seeds from all selected lineswere tested for Bt expression. Twelve BC₃ F₄ progeny were selected fromfour BC₃ F₄ lines [for a selection pressure row of 0.017 (4/241) or 1.7%and a selection pressure plant of 0.003 (12/3600) or 0.03%].

The 12 BC₃ F₄ selections were planted in single row plots replicatedtwice. Each plot was thinned to one plant per foot to facilitate singleplant selection. Tissue from 20 plants per plot were tested for Btexpression to confirm the Bt gene was homozygous. As described above,selection was against all plants exhibiting any upland cotton traits.

At harvest, strong selection pressure was placed on yield. Only fourlines appeared to have yield required for commercial release. Ten plantselections were made per selected line and the remainder of the fourlines were harvested individually [for a 1996 selection pressure row of0.333 (4/12) or 33.3%/]. The bulked lines appeared very homogeneous. Theten selected plants in each line were fiber tested. The fiber qualityaverages were used to determine the quality of the bulked rowselections.

In 1997, the four 1996 bulked lines were entered in two replicatedtests, one in Casa Grande, Ariz. and the other in Maricopa, Ariz.; bothare low elevation desert environments. The four 1996 bulked strains wereincreased on about 0.06 acres [for a final selection pressure plant of0.00056 (2/3600) and row of 0.0041 (1/241)].

The BC₃ F₅ plant selections were planted in single row plots replicatedtwice. Each plot was thinned to one plant per foot to facilitate singleplant selection. Tissue from 20 plants per plot were tested for Btexpression to confirm the plant selections Bt gene was homozygous forthe Bt gene. Fourteen lines were bulk harvested for increase andreplicated testing in 1998.

926 B Pima has superior lint yields to cultivars of similar maturity andadaptation type. It has good agronomic characteristics, including Pimafiber quality..

The criteria used to select in various generations include: lint yield,lint percent, fiber characteristics, maturity, storm resistance andinsect resistance.

The cultivar has shown uniformity and stability to the traits, asdescribed in the following variety description information. It has beenadvanced a sufficient number of generations with carefull attention touniformity of plant type. The line has been increased with continuedobservation for uniformity.

Cotton cultivar 926 B Pima has the following morphologic and othercharacteristics.

Variety Description Information

    ______________________________________                                        Species            Gossypium barbadense L.                                    Areas of Adaptation                                                                              Western United States, Arizona,                                               San Joaquin, New Mexico                                    General                                                                       Plant Habit        Spreading                                                  Foliage            Intermediate                                               Stem Lodging       Intermediate                                               Fruiting Branch    Normal                                                     Growth             Indeterminate                                              Leaf Color         Dark green                                                 Boll Shape         Length more than width                                     Boll Breadth       Broadest at base                                           Maturity                                                                      Date of 50% open bolls                                                                           Undetermined                                               Plant                                                                         1st Fruiting Branch                                                                              Unknown                                                    (from cotyledonary node)                                                      No. Nodes to 1st Fruiting Branch                                                                 8.75                                                       (Excluding cotyledonary node)                                                 Mature Plant Height                                                                              1.55 meters                                                (from cotyledonary node to terminal)                                          Leaf                                                                          (Upper most, fully expanded leaf)                                             Type               Normal                                                     Pubescence         Moderate                                                   Nectaries          Present                                                    Glands                                                                        Leaf               Present                                                    Stem               Present                                                    Calyx Lobe         Present                                                    Flower                                                                        Petals             Yellow                                                     Pollen             Yellow                                                     Petal Spot         Present                                                    Seed                                                                          Seed Index (g/100, fuzzy basis)                                                                  11.6                                                       Boll                                                                          Lint Percent - Picked                                                                            37.6                                                       Number of Seeds per Boll                                                                         14.2                                                       Boll Weight         3.9                                                       Number of Locules per Boll                                                                        3                                                         Boll Type          Open                                                       Fiber Properties                                                              Method             Standard Instrument                                        Length              1.35 inches                                               Uniformity         49.0%                                                      Strength (T1)      32.7 g/tex                                                 Elongation (E1)     8.0%                                                      Micronaire          3.9                                                       Diseases                                                                      Fusarium Wilt      Unknown                                                    Verticillium Wilt  Good resistance                                            Nematodes, Insects and Pests                                                  Root-Knot Nematode Unknown                                                    Boll Weevil        Unknown                                                    Bollworm           Moderately resistant                                       Reniform Nematode  Unknown                                                    Lygus              Moderately resistant                                       Pink Bollworm      Resistant                                                  Tobacco Bud Worm   Resistant                                                  ______________________________________                                    

Deposit Information

A deposit of the cotton seed of 926 B Pima was made with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va. 20110on Jul. 15, 1998 and having ATCC Accession No. 203065.

The cultivar 926 B Pima is similar to Pima S6. While similar, there arenumerous significant differences including: 926 B Pima has a higher seedcotton weight, higher yield, lower micronaire, longer fiber, lower fiberuniformity, lower fiber elongation, taller plant type and smaller seedthan Pima S6. In addition to the yield of the present invention, is thepresence of the Bacillus thuringiensis (Bt) construct 531 gene. Nocommercially available Pima variety carries the Bt gene. Results of pinkbollworm damage are shown in Tables 3 and 4 and results of comparativehead-to-head testing are shown in Table 5.

                  TABLE 3                                                         ______________________________________                                        Pink Bollworm Damage in Bt and Conventional Pima.sup.1                        Percent of hard green bolls with pink bollworm exit holes                     Entry   Rep. 1    Rep. 2  Rep. 3  Rep. 4                                                                              Avg.                                  ______________________________________                                        Pima S-7                                                                              6         --      --      --    --                                    Pima S-6                                                                              16        10      30      10    16                                    926 B Pima                                                                            0          0       0       0     0                                    ______________________________________                                         .sup.1 Sampled 50 hard green bolls per plot. Inspected each boll for exit     holes. Cracked only those bolls with exit holes to verify pink bollworm       damage. Maricopa, Arizona on August 12, 1997.                            

                  TABLE 4                                                         ______________________________________                                        Pink Bollworm Damage in Bt and Conventional Pima.sup.1                        Percent of hard green bolls with pink bollworm exit holes                     Entry     Rep. 1   Rep. 2  Rep. 3  Rep. 4                                                                              Avg.                                 ______________________________________                                        Pima S-6  12       20      36      16    21                                   926 B Pima                                                                              0        0       0       0     0                                    PS6531-328-6B                                                                           0        0       0       0     0                                    930 B Pima                                                                              0        0       0       0     0                                    PS6531-332-6B                                                                           0        0       0       0     0                                    ______________________________________                                         .sup.1 Sampled 25 bolls per plot. Hard green, unopened bolls from the top     portion of the plants were chosen. Inspected each boll for larvae and/or      exit holes. Maricopa, Arizona on September 16, 1997                      

                                      TABLE 5                                     __________________________________________________________________________    1997 Head to Head Comparison                                                        Seed                                                                              Lint                                                                             Lint               Plt                                                                              Mat                                                                              Seed                                    Name  Cotton                                                                            %  Yield                                                                             Mic                                                                              Len                                                                              Ur T1 E1 Hgt                                                                              Rtg                                                                              Index                                   __________________________________________________________________________    Casa Grande, Arizona - 7CGMC138                                               926 B Pima                                                                          4190                                                                              37.7                                                                             1578                                                                              3.6                                                                              1.34                                                                             49.8                                                                             31.4                                                                             8.2                                                                              5.0                                                                              4.5                                                                              11.2                                    Conquistador                                                                        4126                                                                              36.0                                                                             1483                                                                              3.9                                                                              1.34                                                                             50.3                                                                             33.1                                                                             8.3                                                                              2.8                                                                              3.0                                                                              12.2                                    Pima S-7                                                                            3970                                                                              36.5                                                                             1447                                                                              3.9                                                                              1.34                                                                             50.5                                                                             32.6                                                                             7.9                                                                              2.8                                                                              3.0                                                                              12.4                                    Pima S-6                                                                            3519                                                                              37.7                                                                             1325                                                                              3.9                                                                              1.29                                                                             50.3                                                                             31.6                                                                             8.7                                                                              3.5                                                                              4.5                                                                              13.0                                    Average                                                                             3928                                                                              37.2                                                                             1459                                                                              3.8                                                                              1.32                                                                             49.8                                                                             32.3                                                                             8.3                                                                              4.0                                                                              4.1                                                                              12.0                                    LSD (.05)                                                                           131 0.4                                                                              82  0.1                                                                              ns ns ns ns 0.8                                                                              0.2                                                                              0.5                                     Cv (%)                                                                              3.9 0.7                                                                              3.8 1.9                                                                              2.5                                                                              1.7                                                                              2.4                                                                              4.6                                                                              12.8                                                                             10.9                                                                             3.1                                     Reps  4   4  4   4  4  4  4  4  4  4  4                                       Maricopa, Arizona                                                             926 B Pima                                                                          3854                                                                              37.6                                                                             1448                                                                              4.1                                                                              1.36                                                                             48.3                                                                             34.1                                                                             7.7                                                                              4.1                                                                              4.3                                                                              12.0                                    Conquistador                                                                        3621                                                                              37.3                                                                             1351                                                                              4.1                                                                              1.30                                                                             48.4                                                                             34.6                                                                             8.0                                                                              3.1                                                                              2.8                                                                              11.7                                    Pima S-7                                                                            3535                                                                              38.2                                                                             1349                                                                              4.1                                                                              1.33                                                                             49.3                                                                             36.2                                                                             7.2                                                                              2.8                                                                              2.3                                                                              11.7                                    Pima S-6                                                                            3157                                                                              38.4                                                                             1212                                                                              4.1                                                                              1.32                                                                             49.4                                                                             33.8                                                                             8.1                                                                              3.8                                                                              4.0                                                                              12.8                                    Average                                                                             3542                                                                              37.9                                                                             1340                                                                              4.1                                                                              1.32                                                                             48.8                                                                             34.7                                                                             7.7                                                                              3.4                                                                              3.3                                                                              12.0                                    LSD (.05)                                                                           211 0.5                                                                              80  ns 2.62                                                                             0.7                                                                              1.1                                                                              0.3                                                                              0.7                                                                              0.8                                                                              0.6                                     CV (%)                                                                              3.9 0.8                                                                              3.9 2.3                                                                              1.3                                                                              1.0                                                                              2.1                                                                              3.0                                                                              11.7                                                                             13.6                                                                             3.4                                     Reps  4   4  4   4  4  4  4  4  4  4  4                                       Two Location Summary                                                          926 B Pima                                                                          4022                                                                              37.6                                                                             1513                                                                              3.9                                                                              1.35                                                                             49.0                                                                             32.7                                                                             8.0                                                                              4.6                                                                              4.4                                                                              11.6                                    Conquistador                                                                        3874                                                                              36.6                                                                             1417                                                                              4.0                                                                              1.32                                                                             49.3                                                                             33.9                                                                             8.1                                                                              2.9                                                                              2.9                                                                              12.0                                    Pima S-7                                                                            3754                                                                              37.3                                                                             1399                                                                              4.0                                                                              1.33                                                                             50.0                                                                             34.3                                                                             7.5                                                                              2.7                                                                              2.8                                                                              12.0                                    Pima S-6                                                                            3338                                                                              38.0                                                                             1268                                                                              4.0                                                                              1.30                                                                             49.8                                                                             32.7                                                                             8.4                                                                              3.7                                                                              4.3                                                                              12.9                                    Average                                                                             3747                                                                              37.4                                                                             1399                                                                              4.0                                                                              1.32                                                                             49.5                                                                             33.4                                                                             8.0                                                                              3.4                                                                              3.6                                                                              12.i                                    LSD (.05)                                                                           180 0.5                                                                              66  0.1                                                                              0.03                                                                             0.7                                                                              0.8                                                                              0.3                                                                              0.5                                                                              0.5                                                                              0.5                                     CV (%)                                                                              4.7 1.4                                                                              4.7 2.4                                                                              2.1                                                                              1.4                                                                              2.5                                                                              3.8                                                                              12.9                                                                             12.5                                                                             3.9                                     Reps  8   8  8   8  8  8  8  8  8  8  8                                       __________________________________________________________________________     Seed Cotton  Seed cotton pounds/acre                                          Lint %  Lint percent                                                          Lint Yield  Lint yield pounds/acre                                            MIC  Micronaire                                                               Len  2.5% Span length (inches)                                                UR  Uniformity ratio percent                                                  TI  Fiber strength (gr/tex)                                                   E1  Fiber elongation                                                          Ph  Plant height rating (1 = short, 5 = very tall)                            Mat Rtg  Maturity rating                                                      Seed Index  Seed Index (g/100)                                           

EXAMPLE 2 Cotton Cultivar 930 B Pima

In the spring of 1995, seed from 241 individual BC₃ F₂ plants developedat Scott, Miss. were sent to Casa Grande, Ariz. to be planted in thefield for evaluation. Prior to planting, 10 seeds from each individualline were evaluated for Bt expression in the seed. The object was toidentify the lines homozygous for the Bt gene. 65 BC₃ F₃ tested 100% forthe Bt gene and were planted in the field in 1995. Seed from each linewas planted in as many rows as possible and thinned to one plant perfoot to maximize the number of plants for selection.

Throughout the 1995 growing season plants exhibiting any upland cottontraits were tagged so they could be avoided at harvest. Examples oftraits selected against are light green plant color, reduced leaf lobe,cream petal color, absent or no petal spot, cream/intermediate pollencolor, locule numbers different than three, small gossypol glands, ovateshaped bolls and shallow boll pitting.

In June, 1995 ten plants from each row were sampled for Bt expression inplant tissue. It was observed that some plants exhibited weakexpression. The reason is not known, it may be technique or true weakexpression of the gene in specific genotypes. Any row expressing lessthan 100% Bt was discarded. Strong negative selection pressure was alsoplaced on rows weakly expressing the Bt gene.

At harvest each row was visually graded for yield. Once the best lineswere identified each plant in the selected line was examined by hand forfiber quality and naked seed. Substandard plants were discarded.

Selected plants were ginned and the seed was examined for nakedness,lines considered too fuzzy were discarded. The fiber of the remainingplants was tested with standard instruments. All selections exhibitingsubstandard fiber were discarded. 20 seeds from all selected lines weretested for Bt expression. Twelve lines were advanced to the BC₃ F₄generation.

The 12 BC₃ F₄ selections were planted in single row plots replicatedtwice. Each plot was thinned to one plant per foot to facilitate singleplant selection. Tissue from 20 plants per plot were tested for Btexpression to confirm the Bt gene was homozygous. As described above,all plants exhibiting any upland cotton traits were selected against.

At harvest, strong selection pressure was placed on yield. Only fourlines appeared to have yield required for commercial release. Ten plantselections were made per selected line and the remainder of the fourlines were harvested individually. The bulked lines appeared veryhomogeneous. The ten selected plants in each line were fiber tested withstandard instruments. The fiber quality averages were used to determinethe quality of the bulked row selections.

In 1997, the four 1996 bulked lines were entered in two replicatedtests, one in Casa Grande, Ariz. and the other in Maricopa, Ariz.; bothare low elevation desert environments. The four 1996 bulked strains wereincreased on about 0.06 acres.

The BC₃ F₅ plant selections were planted in single row plots replicatedtwice. Each plot was thinned to one plant per foot to facilitate singleplant selection. Tissue from 20 plants per plot were tested for Btexpression to confirm the Bt gene was homozygous. Fourteen lines werebulk harvested for increase and replicated testing in 1998.

930 B Pima has superior lint yields to cultivars of similar maturity andadaptation type. It has good agronomic characteristics, including Pimafiber quality.

The criteria used to select in various generations include: lint yield,lint turnout, fiber characteristics, maturity, storm resistance, diseasetolerance, early season vigor.

The cultivar has shown uniformity and stability to the traits, asdescribed in the following variety description information. It has beenadvanced a sufficient number of generations with carefull attention touniformity of plant type. The line has been increased with continuedobservation for uniformity.

Cotton cultivar 930 B Pima has the following morphologic and othercharacteristics.

Variety Description Information

    ______________________________________                                        Species            Gossypium barbadense L.                                    Areas of Adaptation                                                                              Western United States, Arizona,                                               San Joaquin, New Mexico                                    General                                                                       Plant Habit        Spreading                                                  Foliage            Intermediate                                               Stem Lodging       Intermediate                                               Fruiting Branch    Normal                                                     Growth             Indeterminate                                              Leaf Color         Dark green                                                 Boll Shape         Length more than width                                     Boll Breadth       Broadest at middle                                         Maturity                                                                      Date of 50% open bolls                                                                           Undetermined                                               Plant                                                                         1st Fruiting Branch                                                                              Unknown                                                    (from cotyledonary node)                                                      No. Nodes to 1st Fruiting Branch                                                                  9                                                         (Excluding cotyledonary node)                                                 Mature Plant Height                                                                               1.5 meters                                                (from cotyledonary node to terminal)                                          Leaf                                                                          (Upper most, fully expanded leaf)                                             Type               Normal                                                     Pubescence         Moderate                                                   Nectaries          Present                                                    Glands                                                                        Leaf               Present                                                    Stem               Present                                                    Calyx Lobe         Present                                                    Flower                                                                        Petals             Yellow                                                     Pollen             Yellow                                                     Petal Spot         Present                                                    Seed                                                                          Seed Index (g/100, fuzzy basis)                                                                  11.7                                                       Boll                                                                          Lint Percent - Picked                                                                            38.9                                                       Number of Seeds per Boll                                                                         14.9                                                       Grams Seed Cotton per Boll                                                                        4                                                         Number of Locules per Boll                                                                        3                                                         Boll Type          Open                                                       Fiber Properties                                                              Method             Standard Instrument                                        Length              1.32 inches                                               Uniformity         48.4%                                                      Strength (T1)      33.2 g/tex                                                 Elongation (E1)     8.2%                                                      Micronaire          3.9                                                       Diseases                                                                      Fusarium Wilt      Unknown                                                    Verticillium Wilt  Good resistant                                             Nematodes, Insects and Pests                                                  Root-Knot Nematode Unknown                                                    Boll Weevil        Unknown                                                    Bollworm           Moderately Resistant                                       Reniform Nematode  Unknown                                                    Lygus              Moderately Resistant                                                          (compared to Upland cotton)                                Pink Bollworm      Resistant                                                  Tobacco Bud Worm   Resistant                                                  ______________________________________                                    

Deposit Information

A deposit of the cotton seed of 930 B Pima was made with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va. 20110on Jul. 15, 1998 and having ATCC Accession No. 203066.

The cultivar 930 B Pima is similar to Pima S6. While similar, there arenumerous differences including: 930 B Pima has higher seed cottonweight, higher yield, lower micronaire, longer fiber, lower fiberuniformity, lower fiber elongation, taller plant type and smaller seedthan Pima S6. In addition to the yield of the instant invention, is thepresence of the Bacilus thuringiensis at) construct 531 gene. Nocommercially available Pima variety carries the Bt gene. Results of pinkbollworm damage are shown in Tables 6 and 7 and results of comparativehead-to-head testing are shown in Table 8.

                  TABLE 6                                                         ______________________________________                                        Pink Bollworm Damage in Bt and Conventional Pima.sup.1                        Percent of hard green bolls with pink bollworm exit holes                     Entry   Rep. 1    Rep. 2  Rep. 3  Rep. 4                                                                              Avg.                                  ______________________________________                                        Pima S-7                                                                              6         --      --      --    --                                    Pima S-6                                                                              16        10      30      10    16                                    930 B Pima                                                                            0          0      0        0     0                                    ______________________________________                                         .sup.1 Sampled 50 hard green bolls per plot. Inspected each boll for exit     holes. Cracked only those bolls with exit holes to verify pink bollworm       damage. Maricopa, Arizona on August 12, 1997.                            

                  TABLE 7                                                         ______________________________________                                        Pink Bollworm Damage in Bt and Conventional Pima.sup.1                        Percent of hard green bolls with pink bollworm exit holes                     Entry     Rep. 1   Rep. 2  Rep. 3  Rep. 4                                                                              Avg.                                 ______________________________________                                        Pima S-6  12       20      36      16    21                                   926 B Pima                                                                              0        0       0       0     0                                    PS6531-328-6B                                                                           0        0       0       0     0                                    930 B Pima                                                                              0        0       0       0     0                                    PS6531-332-6B                                                                           0        0       0       0     0                                    ______________________________________                                         .sup.1 Sampled 25 bolls per plot. Hard green, unopened bolls from the top     portion of the plants were chosen. Inspected each boll for larvae and/or      exit holes. Maricopa, Arizona on September 16, 1997.                     

                                      TABLE 8                                     __________________________________________________________________________    1997 Head to Head Comparison                                                        Seed                                                                              Lint                                                                             Lint               Plt                                                                              Mat                                                                              Seed                                    Name  Cotton                                                                            %  Yield                                                                             Mic                                                                              Len                                                                              Ur T1 E1 Hgt                                                                              Rtg                                                                              Index                                   __________________________________________________________________________    Casa Grande, Arizona - 7CG5313                                                930 B Pima                                                                          3956                                                                              38.0                                                                             1502                                                                              3.8                                                                              1.32                                                                             48.8                                                                             32.8                                                                             8.5                                                                              3.8                                                                              3.8                                                                              11.9                                    Conquistador                                                                        4126                                                                              36.0                                                                             1483                                                                              3.9                                                                              1.34                                                                             50.3                                                                             33.1                                                                             8.3                                                                              2.8                                                                              3.0                                                                              12.2                                    Pima S-7                                                                            3970                                                                              36.5                                                                             1447                                                                              3.9                                                                              1.34                                                                             50.5                                                                             32.6                                                                             7.9                                                                              2.8                                                                              3.0                                                                              12.4                                    Pima S-6                                                                            3519                                                                              37.7                                                                             1325                                                                              3.9                                                                              1.29                                                                             50.3                                                                             31.6                                                                             8.7                                                                              3.5                                                                              4.5                                                                              13.0                                    Average                                                                             3928                                                                              37.2                                                                             1459                                                                              3.8                                                                              1.32                                                                             49.8                                                                             32.3                                                                             8.3                                                                              4.0                                                                              4.1                                                                              12.0                                    LSD (.05)                                                                           131 0.4                                                                              82  0.1                                                                              ns ns ns ns 0.8                                                                              0.2                                                                              0.5                                     Cv (%)                                                                              3.9 0.7                                                                              3.8 19 2.5                                                                              1.7                                                                              2.4                                                                              4.6                                                                              12.8                                                                             10.9                                                                             3.1                                     Reps  4   4  4   4  4  4  4  4  4  4  4                                       Maricopa, Arizona                                                             930 B Pima                                                                          3953                                                                              38.9                                                                             1539                                                                              4.1                                                                              1.33                                                                             48.1                                                                             33.7                                                                             7.9                                                                              3.8                                                                              4.3                                                                              11.5                                    Conquistador                                                                        3621                                                                              37.3                                                                             1351                                                                              4.1                                                                              1.30                                                                             48.4                                                                             34.6                                                                             8.0                                                                              3.1                                                                              2.8                                                                              11.7                                    Pima S-7                                                                            3535                                                                              38.2                                                                             1349                                                                              4.1                                                                              1.33                                                                             49.3                                                                             36.2                                                                             7.2                                                                              2.8                                                                              2.3                                                                              11.7                                    Pima S-6                                                                            3157                                                                              38.4                                                                             1212                                                                              4.1                                                                              1.32                                                                             49.4                                                                             33.8                                                                             8.1                                                                              3.8                                                                              4.0                                                                              12.8                                    Average                                                                             3566                                                                              38.2                                                                             1362                                                                              4.1                                                                              1.32                                                                             48.8                                                                             34.6                                                                             7.8                                                                              3.4                                                                              3.3                                                                              11.9                                    LSD (.05)                                                                           21 1                                                                              0.5                                                                              80  ns 2.62                                                                             0.7                                                                              1.1                                                                              0.3                                                                              0.7                                                                              0.8                                                                              0.6                                     CV (%)                                                                              3.9 0.8                                                                              3.9 2.3                                                                              1.3                                                                              1.0                                                                              2.1                                                                              3.0                                                                              11.7                                                                             13.6                                                                             3.4                                     Reps  4   4  4   4  4  4  4  4  4  4  4                                       Two Location Summary                                                          930 B Pima                                                                          3954                                                                              38.5                                                                             1520                                                                              3.9                                                                              1.32                                                                             48.4                                                                             33.2                                                                             8.2                                                                              3.8                                                                              4.0                                                                              11.7                                    Conquistador                                                                        3874                                                                              36.6                                                                             1417                                                                              4.0                                                                              1.32                                                                             49.3                                                                             33.9                                                                             8.1                                                                              2.9                                                                              2.9                                                                              12.0                                    Pima S-7                                                                            3754                                                                              37.3                                                                             1399                                                                              4.0                                                                              1.33                                                                             50.0                                                                             34.3                                                                             7.5                                                                              2.7                                                                              2.8                                                                              12.0                                    Pima S-6                                                                            3338                                                                              38.0                                                                             1268                                                                              4.0                                                                              1.30                                                                             49.8                                                                             32.7                                                                             8.4                                                                              3.7                                                                              4.3                                                                              12.9                                    Average                                                                             3730                                                                              37.6                                                                             1401                                                                              4.0                                                                              1.32                                                                             49.4                                                                             33.5                                                                             8.0                                                                              3.3                                                                              3.5                                                                              12.1                                    LSD (.05)                                                                           180 0.5                                                                              66  0.1                                                                              0.03                                                                             0.7                                                                              0.8                                                                              0.3                                                                              0.5                                                                              0.5                                                                              0.5                                     CV (%)                                                                              4.7 1.4                                                                              4.7 2.4                                                                              2.1                                                                              1.4                                                                              2.5                                                                              3.8                                                                              12.9                                                                             12.5                                                                             3.9                                     Reps  8   8  8   8  8  8  8  8  8  8  8                                       __________________________________________________________________________     Seed Cotton  Seed cotton pounds/acre                                          Lint %  Lint percent                                                          Lint Yield  Lint yield pounds/acre                                            MIC  Micronaire                                                               Len  2.5% Span length (inches)                                                UR  Uniformity ratio percent                                                  E1  Fiber elongation                                                          TI  Fiber strength (gr/tex)                                                   Ph  PIant height rating (1 = short, 5 = very tall)                            Mat Rtg  Maturity rating                                                      Seed Index  Seed Index (g/100)                                           

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

BIBLIOGRAPHY

Allard, R. W., Principles of Plant Breeding, John Wiley & Sons, NewYork, London (1960)

Fehr, W. R., Principles of Cultivar Development, Vol. 2 MacmillanPublishing Co., New York, N.Y., (1987)

Mayo, O., The Theory of Plant Breeding, 2nd Ed., Clarendon Press,Oxford, (1987)

Welsh, J. R. Fundamentals of Plant Genetics and Plant Breeding, JohnWiley & Sons, New York, N.Y., (1981)

What is claimed is:
 1. A cotton cultivar seed designated 930 B Pimahaving ATCC Accession No.
 203066. 2. A plant or its parts produced bygrowing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. Anovule of the plant of claim
 2. 5. A cotton plant having thephysiological and morphological characteristics of the plant of claim 2.6. Tissue culture of the plant of claim
 2. 7. A cotton plant regeneratedfrom the tissue culture of claim 6, wherein said cotton plant is capableof expressing all the physiological and morphological, characteristicsof cotton cultivar 930B.
 8. A method for producing a cotton cultivarseed comprising crossing a first cultivar parent cotton plant with asecond cultivar parent cotton plant and harvesting the resultantcultivar cotton seed, wherein said first or second parent cotton plantis the cotton plant of claim
 2. 9. A hybrid seed produced by the methodof claim
 8. 10. A hybrid plant or its parts produced by growing saidhybrid cotton seed of claim
 9. 11. Seed produced from said hybrid plantof claim
 10. 12. A method for producing a hybrid cotton seed comprisingcrossing a plant according to claim 2 with another, different cottonplant.
 13. A hybrid seed produced by the method of claim
 12. 14. Ahybrid plant or its parts produced by growing said hybrid cotton seed ofclaim
 13. 15. Seed produced from said hybrid plant of claim 14.